Single-spore elemental analyses indicate that dipicolinic acid-deficient Bacillus subtilis spores fail to accumulate calcium

Dipicolinic acid (pyridine-2,6-carboxylic acid; DPA) is a major component of bacterial spores and has been shown to be an important determinant of spore resistance. In the core of dormant Bacillus subtilis spores, DPA is associated with divalent calcium in a 1:1 chelate (Ca–DPA). Spores excrete Ca–DPA during germination, but it is unknown whether Ca and DPA are imported separately or together into the developing spore. Elemental analysis by scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM–EDS) of wild-type spores and mutant spores lacking the ability to synthesize DPA showed that DPA-less spores also lacked calcium, suggesting that the two compounds may be co-imported.     

Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments

Endospore-forming bacteria make up an important and numerically significant component of microbial communities in a range of settings including soils, industry, hospitals and marine sediments extending into the deep subsurface. Bacterial endospores are non-reproductive structures that protect DNA and improve cell survival during periods unfavourable for bacterial growth. An important determinant of endospores withstanding extreme environmental conditions is 2,6-pyridine dicarboxylic acid (i.e. dipicolinic acid, or DPA), which contributes heat resistance. This study presents an improved HPLC-fluorescence method for DPA quantification using a single 10-min run with pre-column Tb³⁺ chelation. Relative to existing DPA quantification methods, specific improvements pertain to sensitivity, detection limit and range, as well as the development of new free DPA and spore-specific DPA proxies. The method distinguishes DPA from intact and recently germinated spores, enabling responses to germinants in natural samples or experiments to be assessed in a new way. DPA-based endospore quantification depends on accurate spore-specific DPA contents, in particular, thermophilic spores are shown to have a higher DPA content, meaning that marine sediments with plentiful thermophilic spores may require spore number estimates to be revisited. This method has a wide range of potential applications for more accurately quantifying bacterial endospores in diverse environmental samples.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

On the isolation of dipicolinic acid from endospores of Sarcina ureae

Summary The isolation of an ultraviolet absorbing material from spores of Sarcina ureae and its identification as dipicolinic acid (DPA) is described. The DPA was present in amounts ranging from 4–7% of the spore dry weight, but was not detected in vegetative cells.The contention that the spores of Sarcina ureae are unusual or atypical, and that the organism is a morphologically aberrant Bacillus species is discussed and found wanting.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.     

Pressure Inactivation of Bacillus Endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

Coordinative binding of divalent cations with ligands related to bacterial spores. Equilibrium studies

It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0°C; μ = 1.0 KNO₃) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), α,ε-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Effects of Lactic Acid Bacteria and Organic Acids on Growth and Germination of Bacillus cereus

Growth and germination of vegetative cells and endospores of Bacillus cereus were affected by Streptococcus lactis, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus in nonfat milk medium and by salts of organic acids in broth medium. Growth of the lactic acid bacteria was not affected by B. cereus. B. cereus increased rapidly to about 10⁸ CFU/ml when cells were added at the beginning of growth of lactic acid bacteria; it was inactivated slowly when added after 24 h and rapidly when added after 72 h of lactic acid bacterial growth. Streptococci were more inhibitory to the growth of B. cereus than lactobacilli were at 24 h. Spore germination was not affected after 24 h, but it was inhibited after 48 and 72 h of lactic acid bacterial growth. Acetate was more inhibitory to the growth of vegetative cells, while formate was more inhibitory to spore germination. Acetate, formate, and lactate (all at 0.1 M) completely inactivated multiplication of B. cereus at pH 6.1, 6.0, and 5.6, respectively. Spores of B. cereus were more resistant to these organic acids compared with the resistance of vegetative cells. Formate, lactate, and acetate (all at 0.1 M) caused 50% inhibition of spore germination at pH 4.4, 4.3, and 4.2, respectively.     

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

Bacterial endospores and their significance in stress resistance

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105°C, 120°C, and 131°C, respectively. The estimated Z values were 6.3°C, 6.1°C, and 9.7°C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (T_c), the temperature at which half the DPA content has been released within a fixed incubation time. We found T_c values for spores from Bacillus strains 168, A163, and IC4 of 108°C, 121°C, and 131°C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.